Cholesterol, triglyceride, and phospholipid are bound to apoproteins in blood plasma to form lipoproteins. In accordance with their physical properties, lipoproteins are classified into, for example, chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). In some cases, LDL is further classified into intermediate density lipoprotein (IDL) and LDL. A product obtained through decomposition of lipoprotein; i.e., a remnant, is occasionally considered a type of lipoprotein. Of these lipoproteins, LDL is known to be a substance that induces arteriosclerosis, whereas HDL is known to exhibit anti-arteriosclerotic activity.
Epidemiologically, the level of apoproteins or cholesterol in LDL is known to exhibit a positive correlation with the frequency of onset of arteriosclerotic diseases, whereas the level of apoproteins or cholesterol in HDL is known to exhibit an inverse correlation with the frequency of onset of arteriosclerotic diseases. Therefore, nowadays, apoproteins or cholesterol contained in LDL or HDL is assayed for the purpose of prevention or diagnosis of ischemic heart diseases.
Known methods for assaying lipids contained in LDL or HDL include a method in which LDL or HDL is separated from other lipoproteins by means of ultracentrifugation, and is then subjected to lipid assay; and a method in which LDL or HDL is separated from other lipoproteins by means of electrophoresis, and is then subjected to lipid staining, followed by measurement of the intensity of coloring. However, these methods have hardly been practiced for problems accompanied by each of the methods such as a requirement for intricate procedures and a difficulty in handling a large number of samples.
Among assays of lipids contained in lipoproteins, HDL cholesterol assays are performed most frequently. In clinical examinations, HDL cholesterol is usually assayed by the precipitation method, in which a precipitant is added to a sample to thereby aggregate lipoproteins other than HDL, then the thus-aggregated lipoproteins are removed through centrifugation, and cholesterol in the thus-separated supernatant containing only HDL is assayed. However, this method requires a relatively large amount of sample, and complete automation of entire analytical steps has not been accomplished. In recent years, there have been proposed a variety of methods that enzymatically separate and assay HDL cholesterol. Examples of such methods include a method employing enzymatic reaction in the presence of a bile salt and a nonionic surfactant (JP-A-63-126498); a method in which lipoproteins other than HDL are aggregated in advance so that HDL cholesterol is only reacted with an enzyme, and subsequently the enzyme is inactivated while the aggregated lipoproteins are re-dissolved simultaneously, followed by measurement of absorbance (JP-A-6-242110); a method employing a precipitant which precipitates lipoproteins other than HDL and a cholesterol assay reagent in combination, in which cholesterol contained in non-precipitated HDL is assayed (JP-B-2600065); a method employing an antibody (JP-A-9-96637); a method employing a sugar compound (JP-A-7-301636); a method in which, in a first reaction, cholesterol oxidase and cholesterol esterase act on lipoproteins other than HDL in the presence of a specific surfactant, to thereby preferentially react these enzymes with cholesterol contained in these lipoproteins, and subsequently HDL cholesterol is assayed while inhibiting any reaction with cholesterol contained in the lipoproteins other than HDL (JP-A-9-299); a method employing a surfactant selected from a specific group and a cholesterol assay enzyme reagent, in which HDL cholesterol is assayed within a period of time when the enzyme reagent is preferentially reacted with the cholesterol contained in HDL (JP-A-11-56395); and a method employing cholesterol oxidase and cholesterol esterase in combination with a surfactant which acts specifically on HDL cholesterol (JP-A-2001-103998).
Clinical significance of an LDL cholesterol assay—which is also commonly performed next to HDL cholesterol—is widely known through large-scale epidemiological studies; however, a method for assaying LDL cholesterol (e.g., a method such as the aforementioned precipitation method for assaying HDL cholesterol) has not been developed. For this reason the LDL cholesterol has been assayed by means of a conversion method (the Freidewald method, hereinafter abbreviated as the “F method”) which calculates an “estimate” on the basis of the results of the ultracentrifugation method. In the case of the F method, the LDL cholesterol level is calculated by subtracting the HDL cholesterol level and the VLDL cholesterol level from the total cholesterol level, where a ⅕ value of the triglyceride level is employed as the VLDL cholesterol level. Since the VLDL cholesterol level is estimated from the triglyceride level, the F method cannot be applied to a patient having a triglyceride level of more than 400 mg/dl or a patient with type III hyperlipidemia. Another problem entailed by the F method is that the assay data are negatively biased when the subject exhibits a transient increase in triglyceride level due to meal intake. In view of the foregoing, there have been developed enzymatic methods for assaying LDL cholesterol, including a method in which HDL cholesterol is eliminated from a sample containing LDL cholesterol, and the remaining LDL cholesterol is assayed (JP-A1-8-828734); and a method in which LDL cholesterol in a sample is assayed in the presence of a sugar compound and/or a protein solubilizing agent (JP-A1-8-829599). Furthermore, there have been proposed a method employing a surfactant having a specific structure (JP-A-9-313200) and a method employing a surfactant which acts on lipoproteins other than LDL in a buffer solution containing an amine (JP-A-10-38888).
In blood plasma, most of the triglyceride exists as VLDL. Therefore, in estimation of the LDL cholesterol level by means of the aforementioned F method, the triglyceride level is employed for estimating the VLDL cholesterol level (VLDL cholesterol level=TG/5). In general, the triglyceride is assayed by means of a method in which free glycerol is consumed in a first reaction, and then free glycerol generated by lipoprotein lipase is phosphorylated in a second reaction, and glycerophosphate oxidase is applied thereon, then the thus-generated hydrogen peroxide is reacted with peroxidase, 4-aminoantipyrine, and a Trinder dye for coloring. Consumption of free glycerol is determined by means of so-called achromatic color appearance method employing either peroxidase and a substrate thereof or catalase, or a combination thereof. Methods conventionally known for assaying triglyceride contained in a specific lipoprotein include a fractionation method employing ultracentrifugation or an aggregating agent; and a fractionation method employing gel filtration. There has also been disclosed a method employing a surfactant which inhibits reaction of lipoproteins other than a specific lipoprotein, or a surfactant having an HLB of 15 or more (International Publication WO 00/43537).